It is a general objective by many scientists working in the field of plant biotechnology to successfully genetically engineer plants of major crop varieties. While plant genetic engineering has been successfully demonstrated in several model plant species, often the model plant species, such as tobacco, carrot and petunia, are not the most economically important plant species for agricultural purposes. Accordingly, much effort has been directed toward the genetic engineering of the more agriculturally important plant species. By the term "genetic engineering" as used herein it is meant to describe the introduction of foreign, often chimeric, genes into one or more plant cells which can be regenerated into whole, sexually competent, viable plants which can be self-pollinated or cross-pollinated with other plants of the same species so that the foreign gene, carried in the germ line, can be inserted into or bred into agriculturally useful plant varieties.
The art of plant tissue culture has been an area of active research for many years but over the past five to ten years an intensified scientific effort has been made to develop regenerable plant tissue culture procedures for the important agricultural crops such as maize, wheat, rice, soybeans, and cotton.
Early publications on tissue culture of cotton dealt mainly with establishing the growing tissues from the plant under aseptic conditions in vitro. Davis, D. G. et al. In Vitro 9:395-398 (1974); Rani, A. and S. S. Bhojwani, Plant Sci. Lett. 7:163-169 (1976); and Price, H. J. et al., Plant Sci. Lett. 10:115-119 (1977). The methods detailed in these publications, however did not provide the necessary framework needed to regenerate cells back into while plants.
In the late 1970's, the development of somatic embryos, i.e., embryos derived from nongametic or somatic tissues, from the wild cotton species G. klotzchianum was reported. Price, H. J. and R. H. Smith, Planta 145:305-307 (1979). Unfortunately, there were two major problems those using the procedure of this report were unable to overcome. First, even after several more years of research, these investigators could not induce the somatic embryos to germinate, i.e., convert, to give rise to whole plants, Finer, J. J. and R. H. Smith, Plant Cell Rep. 3:41-43, 1984. Secondly, the same technique could not be successfully replicated using cultivated cotton as a plant tissue source.
Davidonis and Hamilton were the first to report successful regeneration of whole plants from somatic embryos of cotton. Davidonis, G. H. and R. H. Hamilton, Plant Sci. Lett. 32:89-93 (1984). These experimentors used immature cotyledon tissues of the cultivar Coker 310. The basic medium used consisted of Linsmaier and Skoog (LS) salts, vitamins, and the phytohormones NAA and kinetin. Linsmaier, E. M. and F. Skoog, Physiol. Plant 18:100-127 (1965). The tissues used in this report had been used in culture for several years without reports of similar results, and the exact procedures necessary to replicate this process are still not widely known.
During this same year, the regeneration of plants from several different California cotton cultivars was reported, with a protocol sufficiently developed that it could be replicated by the authors in reasonable time periods. Rangan, T. S. et al., In Vitro 20:256 (1984). In their procedure, several tissues, like cotyledons, immature embryos, and hypocotyl tissue, were cultured on Murashige and Skoog (MS) medium (Murashige, T. and F. Skoog, Physiol. Plant. 15:473-497, 1962) plus the phytohormones NAA and kinetin. After three-to-four months of culture, these tissues gave rise to embryogenic callus and somatic embryos. The embryos were then transferred to a low salt medium, e.g., Beasley and Ting's (BT) medium (Beasley, C. A. and I. P. Ting, Amer. J. Bot. 60:130-139, 1973) plus casein hydrolysate, which permitted some of them to germinate and grow into whole plants. About 200 plants were recovered using the Acala SJ-5 cultivar. Some sterility in the plants was observed and only 2% of the plants showed somaclonal variation.
Somatic embryogenesis was observed using the Coker line 312 and a Texas race stock called T25. Robacker, D. C. and T. W. Zimmerman, In the Ann. Mtg. of the American Society of Agronomy, November 25-30, Las Vegas, Nev. P. 85 (1984). The basal medium consisted of MS salts, the vitamins inositol and thiamine, sucrose, and the phytohormones NAA, 2,4 dichlorophenoxyacetic acid (2,4-D) and kinetin. Hypocotyls were used as the original tissue source. Even though embryos were recovered and cultured onto BT medium, no plants were recovered.
Other investigators have also reported somatic embryogenesis and plant regeneration. (Trolinder, N. L. and J. R. Goodin, In the Proc. of the Beltwide Cotton Production Research Conferences, Jan. 6-11, 1985, New Orleans, La. P. 46; and Mitten, D. H. In the Proc. of the Beltwide Cotton Production Research Conferences, Jan. 6-11, 1985, New Orleans, La. P. 57-58). These procedures were not published in detail, but based on the presentations, enough data was collected such that certain themes began to emerge. One investigator achieved somatic embryogenesis using the Coker 310 cultivar, immature embryos, and hypocotyl tissues on MS medium plus the phytohormones NAA and 2iP (or kinetin). While clear evidence was shown for somatic embryogenesis, the recovery of whole plants from these cultures was less clear. Other experimenters provided a clear and concise protocol. Repeatability was shown as was the recovery of intact plants. While they were able to obtain somatic embryos from several different lines, the best cultivars belonged to the Coker pedigreed lines 5110 and 312 and one Texas race stock, T25. Other cultivars were unable to complete the regeneration process, i.e. convert to whole plants, or were unable to form mature somatic embryos. Basically, their protocol used MS medium, B5 vitamins, and the phytohormones 2,4-D and kinetin.
These investigators have been focusing on plant regeneration of somatic non-transformed cotton tissues, but strategies directed toward the genetic engineering plant lines typically generally involve two complementary processes. The first process involves the genetic transformation of one or more plant cells of a specifically characterized type. By transformation it is meant that a foreign gene, typically a chimeric gene construct, is introduced into the genome of the individual plant cells, typically through the aid of a vector which has the ability to transfer the gene of interest into the genome of the plant cells in culture. The second process then involves the regeneration of the transformed plant cells into whole sexually competent plants. Neither the transformation nor regeneration process need be 100% successful, but must have a reasonable degree of reliability and reproducibility so that a reasonable percentage of the cells can be transformed and regenerated into whole plants.
The two processes, transformation and regeneration, must be complementary. It is possible to transform certain tissues or cell types which cannot be regenerated, and it is also possible to regenerate plant tissues of a number of different tissue and cell types which have not yet been successfully transformed, as demonstrated by the investigators discussed above. The complementarity of the two processes must be such that the tissues which are successfully genetically transformed by the transformation process must be of a type and character, and must be in sufficient health, competency and vitality, so that they can be successfully regenerated into whole plants.
Successful transformation and regeneration techniques have been demonstrated in the prior art for other plant species. For example, in Barton et al., "Regeneration of Intact Tobacco Plants Containing Full-Length Copies of Genetically Engineered T-DNA, and Transmission of DNA to R 1 Progeny", Cell 32:1033 (April 1983), the transformation and regeneration of tobacco plants was reported. Similar results have been achieved in some other plant species, though not cotton.
The most common methodology used for the transformation of cells of dicot plant species involves the use of the plant pathogen Agrobacterium tumefaciens. A. tumefaciens harbors a plasmid, referred to as the tumor-inducing or Ti plasmid, which has the natural ability to transfer a segment of itself, referred to as the T-DNA (transfer-DNA), into the genome of infected plant cells. Wild-type A. tumefaciens use this ability to genetically transform infected cells of plants so that the plant cells become tumorous, and also synthesize one of a series of compounds, known as opines, which can be metabolized by the infecting A. tumefaciens. It has been found by several investigators that by removing the bulk of the T-DNA from a Ti plasmid harbored by A. tumefaciens, and by replacing that T-DNA with a foreign gene construction, that the Agrobacterium can transform infected plant cells with the foreign gene in such a fashion that the resultant cells are not tumorous, as plant cells infected with wild-type normally A. tumefaciens are. The foreign gene construction is then included in the cells of a whole plant regenerated from the transformed cells and is then inherited in a simple Mendelian manner. The construction can thus be treated as any inheritable trait for crop breeding purposes.
Although the regeneration of whole plants from somatic embryos of cotton has been previously reported, it has not been heretofore thought to have been possible to genetically form whole cotton plants utilizing the transformation/regeneration techniques.
It is therefore an object of the present invention to genetically engineer whole intact cotton plants and lines.
It is further an object of the present invention to genetically engineer the production of whole cotton plants utilizing transformation/regeneration techniques.
It is still further an object of the present invention to genetically engineer the production of whole cotton plants, using the Agrobacterium method of gene transformation followed by a reproducible regeneration technique.
These objects and others are fulfilled by the present invention as described below.